Melanins are biopolymers that confer coloration and protection to the host organism against biotic or abiotic insults. The level of protection offered by melanin depends on its biosynthesis and its subcellular localization. Previously, we discovered that Aspergillus fumigatus compartmentalizes melanization in endosomes by recruiting all melanin enzymes to the secretory pathway. Surprisingly, although two laccases involved in the late steps of melanization are conventional secretory proteins, the four enzymes involved in the early steps of melanization lack a signal peptide or a transmembrane domain and are thus considered &ldquo;atypical&rdquo; secretory proteins. In this work, we found interactions among melanin enzymes and all melanin enzymes formed protein complexes. Surprisingly, the formation of protein complexes by melanin enzymes was not critical for their trafficking to the endosomal system. By palmitoylation profiling and biochemical analyses, we discovered that all four early melanin enzymes were strongly palmitoylated during conidiation. However, only the polyketide synthase (PKS) Alb1 was strongly palmitoylated during both vegetative hyphal growth and conidiation when constitutively expressed alone. This posttranslational lipid modification correlates the endosomal localization of all early melanin enzymes. Intriguingly, bioinformatic analyses predict that palmitoylation is a common mechanism for potential membrane association of polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) in A.&nbsp;fumigatus. Our findings indicate that protein-protein interactions facilitate melanization by metabolic channeling, while posttranslational lipid modifications help recruit the atypical enzymes to the secretory pathway, which is critical for compartmentalization of secondary metabolism.

fig1: Interactions between the melanin enzymes. (A) A diagram of the melanin enzymatic pathway that involves four early enzymes and two late enzymes. CoA, coenzyme A. Six strains with Alb1-GFP, Ayg1-GFP, Arp2-GFP, Arp1-GFP, Abr1-GFP, and Abr2-GFP controlled by their native promoters were used in the experiments shown in panels B to D. (B) Melanin enzymes are not produced during vegetative hyphal growth based on the lack of GFP signal (top panel). Scale bar, 5 µm. (C) All 6 melanin enzymes are produced during conidiation based on the strong GFP signals (top panel). Early melanin enzymes are in endosomes, and late enzymes are mostly accumulated in the cell wall. Scale bar, 5 µm. (D) FPLC profiles of Alb1-GFP, Ayg1-GFP, Arp2-GFP, Arp1-GFP, Abr1-GFP, and Abr2-GFP, along with the standard marker protein profiles. The y axis indicates the relative florescence units (RFUs). (E) (a to e) Western blots were probed with GFP antibodies against the protein fraction pulled down by the anti-FLAG antibodies isolated from the Payg1-FLAG+Arp2-GFP, untagged wild-type (WT), and Payg1-FLAG (with only the FLAG tag) control strains. Images from before (a, c, and d) and after (b and e) Co-IP are shown. As expected, no signal was detected from any of the control strains after Co-IP (f, h, j, l, n, and p). The wild-type strain without any tag was used as a negative control (g and i). Signals were detected both before and after Co-IP using the strain with Ayg1-FLAG+Arp2-GFP. Similarly, signals were detected after Co-IP from strains expressing the following tagged proteins: Ayg1-FLAG+Arp1-GFP (k), Abr1-FLAG+Arp2-GFP (m), Abr1-FLAG+Arp1-GFP (o), and Abr1-FLAG+Ayg1-GFP (q).

Mentions:
The effectiveness of protection conferred by melanin depends on its biosynthesis, as well as its subcellular localization (12). In fungi, melanin is found in the cell wall as layers of globular particles, as well as in intracellular and extracellular vesicles (13–17). It is proposed that melanin is synthesized and trafficked through secretory vesicles. However, although some yeast species use classical secretory laccases to polymerize exogenously added precursors for melanization (14), the majority of filamentous fungi synthesize melanin de novo via the polyketide pathway (18). Like other secondary metabolism pathways, melanization through the polyketide pathway involves predicted cytosolic proteins, such as the polyketide synthase (PKS) and modification enzymes, as well as predicted conventional secretory laccases (19). For instance, the dihydroxynaphthalene (DHN) melanin biosynthesis pathway of Aspergillus fumigatus consists of six enzymes encoded by the melanin gene cluster (Fig. 1A). Based on the order of reactions that they carry out, the polyketide synthase Alb1 (also known as PksP) and the modification enzymes Ayg1, Arp1, and Arp2 are categorized as early enzymes, whereas the two laccases Abr1 and Abr2 are categorized as late enzymes (19). We demonstrated recently that the two late enzymes, laccases Abr1 and Abr2, are indeed secretory proteins and that they accumulate in the cell wall of conidiophores and conidia (19, 20). Surprisingly, contrary to the predicted cytoplasmic localization, all four early melanin enzymes are localized to the secretory endosomes (19). The discovery of all melanin enzymes trafficking to/through endosomes provides a plausible explanation for the subcellular compartmentalization of melanin biosynthesis and trafficking in secretory endosomes in Aspergillus.

fig1: Interactions between the melanin enzymes. (A) A diagram of the melanin enzymatic pathway that involves four early enzymes and two late enzymes. CoA, coenzyme A. Six strains with Alb1-GFP, Ayg1-GFP, Arp2-GFP, Arp1-GFP, Abr1-GFP, and Abr2-GFP controlled by their native promoters were used in the experiments shown in panels B to D. (B) Melanin enzymes are not produced during vegetative hyphal growth based on the lack of GFP signal (top panel). Scale bar, 5 µm. (C) All 6 melanin enzymes are produced during conidiation based on the strong GFP signals (top panel). Early melanin enzymes are in endosomes, and late enzymes are mostly accumulated in the cell wall. Scale bar, 5 µm. (D) FPLC profiles of Alb1-GFP, Ayg1-GFP, Arp2-GFP, Arp1-GFP, Abr1-GFP, and Abr2-GFP, along with the standard marker protein profiles. The y axis indicates the relative florescence units (RFUs). (E) (a to e) Western blots were probed with GFP antibodies against the protein fraction pulled down by the anti-FLAG antibodies isolated from the Payg1-FLAG+Arp2-GFP, untagged wild-type (WT), and Payg1-FLAG (with only the FLAG tag) control strains. Images from before (a, c, and d) and after (b and e) Co-IP are shown. As expected, no signal was detected from any of the control strains after Co-IP (f, h, j, l, n, and p). The wild-type strain without any tag was used as a negative control (g and i). Signals were detected both before and after Co-IP using the strain with Ayg1-FLAG+Arp2-GFP. Similarly, signals were detected after Co-IP from strains expressing the following tagged proteins: Ayg1-FLAG+Arp1-GFP (k), Abr1-FLAG+Arp2-GFP (m), Abr1-FLAG+Arp1-GFP (o), and Abr1-FLAG+Ayg1-GFP (q).

Mentions:
The effectiveness of protection conferred by melanin depends on its biosynthesis, as well as its subcellular localization (12). In fungi, melanin is found in the cell wall as layers of globular particles, as well as in intracellular and extracellular vesicles (13–17). It is proposed that melanin is synthesized and trafficked through secretory vesicles. However, although some yeast species use classical secretory laccases to polymerize exogenously added precursors for melanization (14), the majority of filamentous fungi synthesize melanin de novo via the polyketide pathway (18). Like other secondary metabolism pathways, melanization through the polyketide pathway involves predicted cytosolic proteins, such as the polyketide synthase (PKS) and modification enzymes, as well as predicted conventional secretory laccases (19). For instance, the dihydroxynaphthalene (DHN) melanin biosynthesis pathway of Aspergillus fumigatus consists of six enzymes encoded by the melanin gene cluster (Fig. 1A). Based on the order of reactions that they carry out, the polyketide synthase Alb1 (also known as PksP) and the modification enzymes Ayg1, Arp1, and Arp2 are categorized as early enzymes, whereas the two laccases Abr1 and Abr2 are categorized as late enzymes (19). We demonstrated recently that the two late enzymes, laccases Abr1 and Abr2, are indeed secretory proteins and that they accumulate in the cell wall of conidiophores and conidia (19, 20). Surprisingly, contrary to the predicted cytoplasmic localization, all four early melanin enzymes are localized to the secretory endosomes (19). The discovery of all melanin enzymes trafficking to/through endosomes provides a plausible explanation for the subcellular compartmentalization of melanin biosynthesis and trafficking in secretory endosomes in Aspergillus.

Melanins are biopolymers that confer coloration and protection to the host organism against biotic or abiotic insults. The level of protection offered by melanin depends on its biosynthesis and its subcellular localization. Previously, we discovered that Aspergillus fumigatus compartmentalizes melanization in endosomes by recruiting all melanin enzymes to the secretory pathway. Surprisingly, although two laccases involved in the late steps of melanization are conventional secretory proteins, the four enzymes involved in the early steps of melanization lack a signal peptide or a transmembrane domain and are thus considered &ldquo;atypical&rdquo; secretory proteins. In this work, we found interactions among melanin enzymes and all melanin enzymes formed protein complexes. Surprisingly, the formation of protein complexes by melanin enzymes was not critical for their trafficking to the endosomal system. By palmitoylation profiling and biochemical analyses, we discovered that all four early melanin enzymes were strongly palmitoylated during conidiation. However, only the polyketide synthase (PKS) Alb1 was strongly palmitoylated during both vegetative hyphal growth and conidiation when constitutively expressed alone. This posttranslational lipid modification correlates the endosomal localization of all early melanin enzymes. Intriguingly, bioinformatic analyses predict that palmitoylation is a common mechanism for potential membrane association of polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) in A.&nbsp;fumigatus. Our findings indicate that protein-protein interactions facilitate melanization by metabolic channeling, while posttranslational lipid modifications help recruit the atypical enzymes to the secretory pathway, which is critical for compartmentalization of secondary metabolism.